Over the past few years, we have identified and characterized a number of genes encoding novel regulators of centrosome size and centriole duplication. All such szy genes were identified in a screen for factors that genetically interact with the kinase ZYG-1, a conserved upstream regulator of centrosome duplication. Analysis of individual szy genes has led to the identification of several molecular pathways that control centriole duplication by controlling the expression levels of centriole assembly factors. Over the past year we have mainly continued to work on two pathways of particular interest, one of which operates at a post-transcriptional level and a second that operates at a transcriptional level. The first pathway involves the activity of protein phosphatase I (PP1) in negatively regulating centrosome duplication. Loss of either the PP1-&#946; isoform GSP-1 or one of two highly conserved PP1 regulators (named I-2 and SDS-22) suppresses the centriole assembly defect of a zyg-1 hypomorphic mutation. This suggests that PP1 normally opposes the activity of ZYG-1, and accordingly we find a moderate increase in the level of ZYG-1 at centrosomes in embryos compromised for PP1 activity. Furthermore we find that down regulation of PP1 activity results in a three- to five-fold increase in the total cellular levels of ZYG-1 indicating that PP1 functions to limit expression of ZYG-1. As zyg-1 mRNA levels are unaffected by inhibition of PP1 activity, PP1 appears to act post-transcriptionally. Additional experiments performed during the past year indicate that PP1 acts post-translationally. Interestingly, we find that in a zyg-1(+) background, strong down regulation of either I-2 or SDS-22 results in the overproduction of centrioles, the formation of multipolar spindles and ultimately lethality. Using structured illumination microscopy (SIM) we have confirmed the centriole over-duplication defect and have found that more than one daughter forms next to each mother centriole. Currently we are trying to identify the molecular target(s) of PP1. The most obvious candidate is ZYG-1, which contains a consensus PP1-docking motif. We have taken several approaches to determine if PP1 and ZYG-1 physically interact. These include co-immunoprecipitation (co-IP) experiments to determine if ZYG-1 and GSP-1 reside in a complex in vivo and mutating zyg-1 to ablate the docking motif and determine if this affects ZYG-1 protein levels. So far we have not been able to detect an interaction between ZYG-1 and PP1, I-2, or SDS-22. However it is possible that co-IP experiments failed to detect an interaction because it is transient in nature. To address this we have fused PP1 to the biotin ligase birA and ZYG-1 to the biotin-acceptor tag. We plan to co-express these two fusion proteins in the same strain and look to see if ZYG-1 is biotinylated. While our co-IP experiments failed to demonstrate a ZYG-1-PP1 interaction, they did succeed in demonstrating that PP1, I-2 and SDS-22 all interact with each other and with a small RNA-binding protein called CSTL-2. Interestingly, down regulation of CSTL-2 suppresses the centriole assembly defect of a zyg-1 hypomorphic mutation, similar to the effect of down regulating PP1. We are currently trying to determine if CSTL-2 functions in the PP1-depedent regulation of ZYG-1. The second pathway of interest regulates centriole duplication at a transcriptional level. This is an important yet overlooked area of investigation as most studies have focused on post-transcriptional mechanisms of regulation. We have found that in the worm, the heterodimeric transcription factor E2F-DP1 represses centrosome duplication. Specifically we find that loss of E2F or DP1 activity can suppress a zyg-1 hypomorphic allele. Accordingly, conserved binding sites for this transcription factor are found in the promoter regions of most of the centriole duplication genes including zyg-1 and sas-6, and recent ChiP-on-Chip analysis demonstrates that these sites are bound in vivo by E2F-DP1 (Kudron et al. 2013 Genome Biol. 14: R5). We have performed qRT-PCR analysis of transcript levels in wild-type and dpl-1 mutants and find that E2F-DP1 is likely to play a positive role in regulating transcription of centriole duplication genes. Consistent with this, deletion of E2F-binding site in the promoter of ZYG-1 abolishes expression. Despite our finding that E2F-DP1 plays a positive role in regulating expression of centriole duplication genes including SAS-6, we find that loss of E2F-DP1 specifically results in an increase in the level of SAS-6 protein. This seemingly contradictory result can be explained if E2F-DP1 also positively regulates the expression of a gene that down regulates SAS-6 protein levels. Our data thus favors a model whereby E2F-DP1 through its role in transcription sets the balance between positive and negative regulators of centriole duplication and that a partial loss of E2F-DP1 tips the balance in favor of the positive regulators. Finally, we have also begun to characterize another regulator of centriole duplication. The chromodomain helicase CHD-1 is known to positively and negatively regulate transcription. Among its known targets are genes encoding components of the centriole duplication pathway. Recently, CHD-1 was found to be co-precipitated with the ZYG-1 ortholog Plx4 in Xenopus egg extracts (Hatch et al. 2010 JCB 191: 721). We have found that depletion of the worm chd-1 homolog suppresses the centriole duplication defect of the zyg-1(it25) mutant. A mutation in the chd-1 gene can also suppress zyg-1(it25) indicating that chd-1 negatively regulates centriole duplication. While loss of chd-1 function does not affect ZYG-1 or SAS-6 protein levels, the SAS-6 protein in chd-1 mutant worms exhibits altered mobility by SDS-PAGE indicating an altered post-translational modification. We are currently trying to identify this modification and how it might be regulated by CHD-1. In the past year we have developed the method of CRISPR-based genome editing in the worm. We have used this method to cleanly delete the chd-1 gene and replace it with GFP. This has allowed us to explore the consequences of a complete loss of CHD-1 on the worm, while creating a transcriptional reporter to determine where CHD-1 is normally expressed. Interestingly, we find that deletion of the chd-1 gene results in a partial embryonic lethal phenotype. We are currently exploring the basis for this lethality. We have also used CRISPR to tag the chd-1 orf with gfp and have found that GFP-CHD-1 localizes to nuclei in most cells of the worm. Finally we have used siRNAs to knock down chd1 in human tissue culture cells and have found that this causes a modest but reproducible centriole amplification phenotype. Going forward we plan to study how CHD-1 may regulate centriole duplication in both worms and human cells.